The continuous development of industry relates to the appearance in the environment of chemical compounds which under normal conditions do not occur there. At present, nitro compounds are amongst the major types of anthropogenic pollution, among which nitrobenzene, 2- and 4-nitrotoluene, 3-nitrotoluene, 4-nitrophenol, 3-nitrophenol and p-nitroaniline deserve particular attention. They are used in the manufacture of explosives, in pesticide and herbicide production, as substrates in the synthesis of dyes, plastics, paints, as well as in the pharmaceutical industry. It is estimated, that each year about 108 tons of organic nitro compounds are produced in the world, and more than 8.5 tons of nitrobenzene alone are released into the environment. For almost 80 years the widespread use of these compounds in many branches of the industry, as well as the production of massive amounts of military materials and ammunition in the first half of the previous century in connection with the two world wars have contributed to the serious contamination of the environment with nitro compounds. The aforementioned types of compounds and their metabolites are highly toxic and dangerous to humans. Some of them are potent poisons, often with strong mutagenic and carcinogenic properties. Most of the aromatic nitro compounds are characterised by their stability and persistence in biological systems, and their considerable resistance to degradation (Kulkarni and Chaudhari, 2007).
An additional concern is the fact that the contamination of soil with nitro compounds, is also a direct threat to groundwater, and consequently may result in the penetration of these pollutants into flowing waters. The problem of soil and groundwater contamination with organic compounds from various branches of the industry, concerns not only Poland and other European Union countries, but practically all industrialized countries of the world. Both in Poland and worldwide, the problem of environmental pollution with these compounds concerns mainly the areas surrounding chemical plants, where they were used as substrates in organic synthesis, as well as the areas of military bases, where they were kept and stored.
There are several conventional methods of physico-chemical neutralization of nitro compounds, such as oxidation and photo-oxidation, hydrolysis, evaporation, combustion, adsorption, etc. (Kanekar et al., 2003). Such methods, however, have numerous drawbacks and limitations. Combustion is not cost-effective nor environmentally friendly. Additionally, it is accompanied by the release of considerable amounts of toxic fumes into the environment. During such procedures as filtration, extraction or adsorption on resin, the undesirable compounds are only separated, and this does not lead to their breakdown. Oxidation processes, meanwhile, generate the formation of toxic derivatives and are associated with high costs (Kulkarni and Chaudhari, 2007).
Several land remediation strategies, based on physical, chemical and biological methods were developed. It is believed, however, that bioremediation technologies that are the most inexpensive and most effective and thus safe are ones, utilizing the metabolic potential of microorganisms.
Bioremediation is a process of repair, in which microorganisms such as bacteria, yeasts and filamentous fungi are used to decompose hazardous substances to less toxic or non-toxic compounds.
For the purposes of bioremediation processes, microorganisms are isolated from the natural microflora present in the contaminated environment (reinoculation), or are obtained by methods of genetic engineering. In practice, in the biodegradation process, specialized kits (consortia of microorganisms) showing particular abilities to degrade certain groups of hydrocarbons are involved. Such consortia, apart from the high detoxification activity, must quickly adapt to the contaminated environment, cooperate with the indigenous microflora and not to accumulate toxic decomposition intermediates.
Two types of microbiological preparations can be distinguished: preparations containing strains of external origin and autovaccines containing effective indigenous strains, isolated from treated soil using various methods.
Patent PL 180 141 discloses a method of microbiological remediation of petroleum-contaminated soils, in which indigenous microorganisms isolated from soil intended to undergo treatment, are utilized. The isolated strains of bacteria are grown under aerobic conditions in a liquid mineral medium supplemented with petroleum hydrocarbons, and after identification, bacteria with the highest activity in degrading these pollutants are selected. The selected 5 to 10 different species of bacteria, are grown at 26° C. for 48 to 72 hours. The propagated culture is introduced into the contaminated soil, by spraying it with an aqueous suspension of bacteria, with more than 105 cells per 1 g of dry soil.
Patent PL 189 586 discloses the method of preparation of an autovaccine that accelerates the treatment of soil and waste water contaminated with petroleum, which relies on the isolation of bacteria from soil and waste waters through dilution and selective culturing in media enriched with sterile crude oil or naphthalene as the sole carbon source.
Prolonged presence of aromatic nitro compounds, particularly nitroaniline, nitrobenzene and nitrophenol in soil initiates the process of natural adaptation and selection of microorganisms in the contaminated area, which affects the quality, as well as the species composition of autochthonic (native to a given ecosystem) groups of microorganisms. Effective bioremediation requires, apart from the accurate knowledge of the microorganisms responsible for the degradation of given aromatic nitro compounds, the understanding of the degradation pathways of these compounds, at the physiological, biochemical, and molecular levels, as well as research on the optimization of conditions necessary for the smooth running of the bioremediation processes. Such studies have been carried out by the Authors of the present solution.
In soils from military sites (i.a. military training grounds) and industrialized areas, high concentrations of not only a variety of organic xenobiotics, but also of heavy metals such as arsenic, cadmium, chromium, copper, lead, mercury, nickel, zinc and others are very often reported (Bahig and Altalhi, 2009). Heavy metals are considered to be potent inhibitors of organic xenobiotic biodegradation processes (Silva et al., 2007). It is thought that the presence of heavy metals in industrial waste waters is one of the main factors limiting the use of biological remediation methods (Kulkami and Chaudhari, 2007). The long-term presence of these pollutants in the environment, however, resulted in bacteria developing mechanisms of detoxifying these compounds. Furthermore, it is suggested that microbial tolerance to heavy metals can affect the maintenance and passing of antibiotic resistance genes between bacteria, by increasing the selective pressure of the environment (Spain, 2003). There is also evidence of a connection between the resistance of bacteria to many clinically important classes of antibacterial drugs, heavy metals and quaternary ammonium compounds used as disinfectants. In many cases, this is related to the location of genes that determine such resistance, in close proximity on the same bacterial plasmid, suggesting the possibility of passing of whole gene clusters by horizontal gene transfer (Schluter et al., 2007).
There are numerous studies concerning the process of bioremediation of various xenobiotics polluting soil e.g. crude oil and its derivatives, but, at the current state of the art, there are no studies concerning the use of in situ bioremediation of areas contaminated with aromatic nitro compounds. In the professional literature on biodegradation of cyclic nitro compounds, this problem has been considered and analysed only on the laboratory scale. The application of an effective method of nitro compound biodegradation, particularly of nitroaniline, seems to be the optimal solution in many respects, primarily because these are processes that occur naturally in the environment, but also, what is of great importance, they are very efficient and are associated with lower costs than traditional methods such as physico-chemical techniques.
Polish patent PL 380 007 relates to a method of soil bioremediation and prevention of the spreading of contamination with organic substances, based on the introduction of a yeast species Yarrowia lipolytica in an immobilised form into the soil. The yeast are introduced directly into the contaminated areas or around them, using an in situ method in the torr of a vaccine (biological preparation) on an organic medium, which is sodium alginate, agar, gelatine, collagen or bird feathers. The vaccine, in a liquid or dry form, may be introduced as granules or a biofilm or biogel, and the yeast constitute from 5 to 50% of the vaccine (by mass), wherein the vaccine may constitute from 10 to 100% of the material introduced into the soil. The vaccine is placed at a depth of 0.1 to 2 m into holes of a diameter of 0.1 to 1.0 m, whereas their distribution may be linear or transversely-overlapping or concentric or encircling the given area or situated at its lowest point, according to the direction of groundwater flow, and in the case of the protection of water bodies, near the shoreline, beyond the reach of the waves. The invention can find use especially in areas contaminated by petroleum compounds, oil industry wastes, vegetable and mineral oils.
Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.